CN108530281B - Isopimarane diterpene derivative, pharmaceutical composition and application thereof - Google Patents
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Abstract
Isopimarane diterpenoid derivatives (1-7) shown in a structural formula (I), a pharmaceutical composition comprising the derivatives as active ingredients and a medicinal carrier, a preparation method of the isopimane diterpenoid derivatives (1-7) and the pharmaceutical composition thereof, and application of the derivatives in preparing medicaments for treating human diseases, in particular application in preparing medicaments for treating various estrogen-mediated diseases, and application of the derivatives as estrogen biosynthesis regulators.
Description
The technical field is as follows:
the invention belongs to the technical field of medicaments, and particularly relates to isopimarane diterpenoid derivatives, a medicinal composition containing the isopimarane diterpenoid derivatives as an active ingredient and used for treating estrogen-related diseases, in particular to application of the isopimarane diterpenoid derivatives in preparation of medicaments for treating various estrogen-mediated diseases, a preparation method of the isopimarane diterpenoid derivatives, and application of the isopimarane diterpenoid derivatives as estrogen biosynthesis regulators.
Background art:
estrogen is an important endogenous substance in the human body and mediates many important physiological functions. The estrogens are mainly of three types, the highest in activity is estradiol (17 beta-estradiol, E2), the next is estrone (E1), the lowest in activity is estriol (E3), and the biological effect of the estrogens plays an important role in reproduction, immunity, bone, cardiovascular and central nervous systems mainly by binding with estrogen receptor (estrogen receptor alpha/beta) to activate the transcriptional or non-transcriptional activity of the estrogens. Estrogen deficiency often causes common diseases such as osteoporosis, coronary heart disease, alzheimer disease, obesity and the like, and the relative excess of estrogen is a direct reason for the occurrence, development, metastasis and the like of various tumors (such as breast cancer). In organisms, cholesterol synthesizes estrogen through a series of enzymatic reactions, the rate-limiting step of which is the conversion of androgen substrates to estrogen by aromatase. Aromatase is expressed in ovary, adipose tissue and bone, but the regulation mechanism is different when different promoters are used in different tissues.
In China, with the development of society and economy, the morbidity, mortality and risk factors of many senile diseases (such as cardiovascular and cerebrovascular diseases, osteoporosis and breast cancer) are in a straight-line trend in nearly ten years. For example, spine or crotch fractures caused by osteoporosis are a major cause of morbidity and mortality in the elderly. Breast cancer is already the most prevalent malignancy in women, severely affecting the quality of life in postmenopausal women. These diseases are related to the anabolic disorder of estrogen in the body and/or the disorder of its mediated signaling pathway, so that the discovery of novel estrogen synthesis modulators is of great significance for the treatment of these major diseases.
The natural product is a natural small molecular substance structure database due to the diversity of molecular frameworks and substituents of the natural product, and is also an important source for the research and development of modern medicines. The Premna microphylla has effects in promoting blood circulation, dispelling blood stasis, strengthening tendons and bones, expelling pathogenic wind, and relieving pain. In fact, the dried stem of premna microphylla is called as war bone, and has obvious effects of resisting inflammation, reducing swelling, relieving pain, improving microcirculation and protecting sciatic nerve and soft tissue injury. Is often used for treating scapulohumeral periarthritis, hyperosteogeny, hypertrophic spondylitis, lymphadenitis, lumbago, leg pain, and pain of liver region in folks. However, studies on chemical components and biological activities thereof have been rarely reported.
So far, no report of the isopimarane diterpene derivatives (1-7 shown in formula I) and no report of the pharmaceutical compositions of the isopimarane diterpene derivatives as active ingredients exist in the prior art, and no report of the application of the isopimarane diterpene derivatives (1-9 shown in formula I) and the pharmaceutical compositions of the isopimarane diterpene derivatives in the preparation of estrogen biosynthesis regulators and the medicines for treating estrogen-mediated diseases exists.
The invention content is as follows:
the invention aims to provide a novel isopimarane diterpenoid derivative (1-7) shown in a formula (I) with medicinal value, a medicinal composition which contains the isopimarane diterpenoid derivative (1-7) (I) and a medicinal carrier and can be used as an estrogen synthesis regulator and treat estrogen-mediated diseases, a preparation method of the isopimarane diterpenoid derivative (1-7) and the medicinal composition thereof, and application of the compound or the medicinal composition thereof in preparing an estrogen biosynthesis regulator and medicaments for treating estrogen-mediated diseases.
In order to achieve the above purpose of the present invention, the present invention provides the following technical solutions:
an isopimarane diterpene derivative (1-7) shown in a structural formula (I),
the invention also provides a pharmaceutical composition, which comprises a therapeutically effective amount of any one of the isopimarane-type diterpene derivatives (1-7) shown in the formula (I) in claim 1 and a pharmaceutically acceptable carrier.
The invention also provides application of the isopimarane diterpenoid derivative (1-7) shown in the formula (I) in preparation of an estrogen biosynthesis regulator.
The isopimarane diterpenoid derivative (1-7) shown in the formula (I) is applied to the preparation of medicines for treating various diseases mediated by estrogen.
The isopimarane diterpene derivative (1-7) shown in the formula (I) is used as an estrogen biosynthesis regulator.
The application of the pharmaceutical composition in preparing estrogen biosynthesis regulators.
The application of the pharmaceutical composition in preparing medicines for treating various diseases mediated by estrogen.
The pharmaceutical composition is used as an estrogen biosynthesis regulator.
The invention also provides a method for preparing the isopimarane diterpene derivative (1-7) shown in the formula (I), which comprises the steps of taking the leaf and branch parts of the premna fulva, drying, crushing, extracting for 3 times at room temperature by using 80% ethanol, combining ethanol extract, filtering, and concentrating under reduced pressure; suspending the extractive solution in water solution, extracting with ethyl acetate, concentrating the ethyl acetate part under reduced pressure, dissolving with chloroform, adsorbing on silica gel, standing at room temperature for volatilizing solvent, performing silica gel column chromatography, and gradient eluting with chloroform/acetone of 1:0 → 0:1 to obtain five parts A-E; wherein the C part is decolorized with MCI, eluted with 95% methanol/water, concentrated, and then separated again with RP-18 reverse phase silica gel, and eluted with methanol/water gradient to obtain C1-C8 fraction; c4 is further separated by Sephadex LH-20 gel, methanol isocratic elution is carried out, and C4.1-C4.5 distillation segments are obtained; separating the C4.3 part with normal phase silica gel, eluting with petroleum ether/acetone at 8:1-1:1 gradient, separating into C4.3.1-C4.3.8 fractions, recrystallizing compounds 2 and 7 from C4.3.5 and C4.3.7 fractions, further purifying fraction C4.3.4 with HPLC, acetonitrile/water, 48:52, 3mL/min to obtain compounds 3 and 4, separating and purifying C4.4 fraction with HPLC, acetonitrile/water, 46:54, 3mL/min to obtain compounds 1, 5 and 6.
The method for preparing the pharmaceutical composition containing the isopimarane diterpene derivatives (1-7) is to take the isopimarane diterpene derivatives (1-7) as raw materials and add pharmaceutically acceptable carriers or excipients.
When the compound of the present invention is used as a medicament, it may be used as it is or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1-99%, preferably 0.5-90% of the compound of the present invention, the balance being pharmaceutically acceptable, pharmaceutically acceptable carriers and/or excipients that are non-toxic and inert to humans and animals.
The pharmaceutically acceptable carrier or excipient is one or more of solid, semi-solid and liquid diluents, fillers and pharmaceutical adjuvants. The pharmaceutical composition of the present invention is used in the form of a dose per unit body weight. The medicine of the present invention may be administrated through injection and oral taking.
Description of the drawings:
FIG. 1 inhibition of estrogen biosynthesis by Compound 2.
FIG. 2 Compound 3 promotes estrogen biosynthesis.
FIG. 3 the effect of Compound 2 on inhibition of estrogen synthesis as a function of time.
FIG. 4 shows the effect of compound 3 in promoting estrogen synthesis over time.
FIG. 5 Compound 2 inhibits aromatase mRNA production.
FIG. 6 Compound 3 promotes aromatase mRNA production.
FIG. 7. Compound 2 inhibits aromatase production.
FIG. 8 Compound 3 promotes aromatase production.
FIG. 9 is a schematic structural diagram of the isopimarane-type diterpene derivatives (1-7) of the formula (I) of the present invention.
The specific implementation mode is as follows:
in order to better understand the essence of the present invention, the following test examples are provided to illustrate the pharmacological effects of the isopimarane-type diterpene derivatives (1-7) of the present invention with reference to the drawings, but the present invention is not limited thereto.
Test example 1:
isopimarane diterpene derivatives (1-7) of formula (I) have an in vivo estrogen biosynthesis-regulating effect:
1.1 cell-based estrogen biosynthesis assay. KGN cells were seeded overnight in 24-well plates. The following day, the medium was replaced with serum-free DMEM/F-12 medium, the cells were pretreated for 24 hours, then testosterone (10nm) was added to each well, and the cells were cultured for 24 hours. The magnetic particle separation enzyme-linked immunosorbent assay (ELISA kit, Beijing, China) is used for detecting the content of the KGN cell 17 beta-estradiol. The assay results can be expressed as a percentage of the control by normalization to total cellular protein content. Protein detection was determined using the BCA protein detection kit (Pierce, Rockford, IL., USA).
1.2 real-time quantitative PCR. Total cellular RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Total RNA was reverse transcribed using an oligonucleotide (dT)18 primer and SuperScript III reverse transcriptase (Invitrogen). The same amount of complementary DNA was subjected to real-time quantitative PCR using the fluorescent dye SYBR Green I according to the manufacturer's protocol (Fermentas, Thermo Scientific). The following primer pairs were used for aromatase, promoter II3 and GAPDH:
5′-ACCCTTCTGCGTCGTGTC-3′/5′-TCTGTGGAAATCCTGCGTCTT-3′(aromatase sense/antisense),5′-TCCCTTTGATTTCCACAGGACTC-3′/5′-ATGCAGTAGCCAG GACCTGGT-3′(promoter II sense/antisense)；5′-CACTCTACCCACTCAAGGGCA-3′/5′-TTGGCTTGAATTGCAGCATTT-3′(promoter I.3 sense/antisense)；
5′-TGCACCACCAACTGCTTAGC-3′/5′-GGCATGGACTGTGGTCATGAG-3′(GAP-DH sense/antisense)。
the amount of mRNA for aromatase, promoter II and promoter I.3 was normalized to the amount of endogenous reference (GAPDH) mRNA in the same sample.
1.3 Western blot. Cells were lysed in RIPA buffer (Byotime, Haimen, China) plus protease inhibitor (Sigma-Aldrich). Cell lysates (50. mu.g) were subjected to 10% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad, Hercules, Calif., USA). Aromatase antibodies (Epitomics, Burlingame, CA, USA), GAPDH antibodies (Abgent, Suzhou, China), pho-CREB antibodies, pho-ERK antibodies, pho-JNK antibodies (Cell Signaling Technology, Danvers, MA, USA), pho-p38 antibodies, pho-AKT antibodies (Signaling Antibody LLC, College Park, MD, USA). Secondary antibodies conjugated to horseradish peroxidase (Pierce) were used for protein detection. Membranes were developed using enhanced chemiluminescence detection (Amersham Bioscience, Piscataway, NJ, USA). Protein concentration was determined by using BCA protein detection kit (Pierce).
2. And (3) test results:
2.1 through estrogen biosynthesis experiments (results are shown in figures 1-4), compound 2 is found to have significant inhibition of estrogen biosynthesis, IC50The value was 10.68. + -. 0.215. mu.M; while Compound 3 exhibits the opposite (i.e., promoting estrogen biosynthesis), EC50The value was 15.49. + -. 0.148. mu.M. Furthermore, the influence of these two compounds on estrogen biosynthesis is time and dose dependent.
2.2 by real-time quantitative PCR assay (results are shown in FIGS. 5 and 6), significant decrease and increase in the expression level of aromatase mRNA in KGN cells treated with compounds 2 and 3 were observed, and a dose-dependent relationship was shown. The results of western blot assay for detecting aromatase were consistent with those of quantitative PCR assay (the results are shown in fig. 7 and 8), and the amount of aromatase protein in KGN cells treated with compounds 2 and 3 was significantly decreased and increased, and showed a dose-dependent relationship.
3. And (4) conclusion:
the experimental result shows that the compound 2 has obvious effect of inhibiting estrogen biosynthesisEffect of the invention, IC50The value was 10.68. + -. 0.215. mu.M; while Compound 3 shows an effect of promoting estrogen biosynthesis, EC50The value was 15.49. + -. 0.148. mu.M. And both compounds can affect the biological synthesis of estrogen by regulating the expression of mRNA of aromatase.
The preparation method and the pharmaceutical composition of the present invention will be further illustrated by the following examples of the present invention, but the present invention is not limited thereto.
Example 1:
preparation of Isopimarane-type diterpene derivatives (1-7) (I):
extraction and separation of isopimarane diterpene derivatives (1-7) (I):
the semi-preparative HPLC used in the experiment were Agilent 1260 and Zorbax SB-C18(9.4 mm. times.25 cm) columns; thin-layer chromatography silica gel and column chromatography silica gel (100-; the reverse C18 silica gel was Lichroprep RP-18gel (40-63 μm, Merck, Darmstadt, Germany) and the Sephadex LH-20 gel (Pharmacia).
Collecting leaf and branch parts of Premna fulva, drying, pulverizing, extracting with 80% ethanol at room temperature for 3 times, mixing ethanol extractive solutions, filtering, and concentrating under reduced pressure; suspending the extractive solution in water solution, extracting with ethyl acetate, concentrating the ethyl acetate part under reduced pressure, dissolving with chloroform, adsorbing on silica gel, standing at room temperature for volatilizing solvent, performing silica gel column chromatography, and gradient eluting with chloroform/acetone of 1:0 → 0:1 to obtain five parts A-E; wherein the C fraction is decolorized with MCI (95% methanol/water elution), the eluate is concentrated and re-separated with RP-18 reverse phase silica gel (methanol/water gradient elution) to obtain C1-C8 fraction; c4 is further separated by Sephadex LH-20 gel (methanol isocratic elution) to obtain C4.1-C4.5 fraction; the C4.3 fraction was separated by normal phase silica gel separation (petroleum ether/acetone, 8:1-1:1 gradient elution) into C4.3.1-C4.3.8 fractions. Compounds 2 and 7 were recrystallized from C4.3.5 and C4.3.7 fractions, respectively. Fraction C4.4.4 was further purified by HPLC (acetonitrile/water, 48:52, 3mL/min) to afford compounds 3 and 4. Separating and purifying C4.4 fraction by HPLC (acetonitrile/water, 46:54, 3mL/min) to obtainCompounds 1, 5 and 6. The structure of the above compound is as follows1H，13C NMR, IR, UV and mass spectral data were determined.
Structural data of isopimarane-type diterpene derivatives (1-9):
optical rotation was determined by SEPA-300 and Jascomodel 1020 polarimeters (Horiba, Tokyo, Japan); infrared spectroscopy (IR) by KBr pellet method using a Tenor 27 type infrared spectrometer; the ultraviolet spectrum was measured by a UV-2401A ultraviolet spectrometer (Shimadzu); measuring nuclear magnetic resonance spectrum (NMR) by using Brucker AM-400 type and DRX-500 type superconducting nuclear magnetic resonance instruments, taking acetone as a solvent and TMS (tetramethylsilane) as an internal standard; high resolution mass spectrometry (HREI-MS) was determined using an API Qstar Pulsar mass spectrometer. The NMR data of the compounds are shown in tables 1 and 2.
TABLE 1 preparation of isopimarane-type diterpene derivatives (1-7) (I)13C NMR spectral data Retention (600MHz) (delta: ppm)
aThe compounds are represented in pyridine-d5Detecting in a solvent;bthe compound is represented in CD3And detecting in OD solvent.
TABLE 2 methods for producing diterpene derivatives (1-7) (I) of the isopimaric type1H NMR spectral data Retention (600MHz) (delta: ppm, J: Hz)
aThe compounds are represented in pyridine-d5Detecting in a solvent;bthe compounds are represented byCD3And detecting in OD solvent.
The molecular formula is as follows: c20H32O4
Molecular weight: 336.23
The characteristics are as follows: colorless crystals
Optically active
IR(KBr)vmax:3426,2934,1693,1452,1380,1230,1143,1040,907,543cm-1。
UV/Vis(MeOH)λmax(logε):206(3.53)nm。
ESIMS[M+Na]+m/z:359。
HRESIMS[M+Na]+m/z:359.2199(calcd for 359.2198).。
The molecular formula is as follows: c20H32O2
Molecular weight: 304.24
The characteristics are as follows: colorless crystals
Optically active
IR(KBr)vmax:3425,2931,1631,1381,1015,572cm-1。
UV/Vis(MeOH)λmax(logε):208(3.81)nm。
ESIMS[M+Na]+m/z:327。
HRESIMS[M+Na]+m/z:327.2295(calcd for 327.2300)。
The molecular formula is as follows: c20H30O3
Molecular weight: 318.22
The characteristics are as follows: colorless crystals
Optically active
IR(KBr)vmax:3422,2927,1649,1381,1268,1191,1026,615cm-1。
UV/Vis(MeOH)λmax(logε):203(3.66),270(3.73)nm。
ESIMS[M+Na]+m/z:341。
HRESIMS[M+Na]+m/z:341.2097(calcd for 341.2093)。
Compound 4
The molecular formula is as follows: c20H30O3
Molecular weight: 318.22
The characteristics are as follows: colorless crystals
Optically active
IR(KBr)vmax:3421,2928,2861,1650,1461,1383,1207,1037,619cm-1。
UV/Vis(MeOH)λmax(logε):201(3.26),268(3.70)nm。
EIMS[M-H]-m/z:317。
HRESIMS[M+Na]+m/z:341.2094(calcd for 341.2093)。
The molecular formula is as follows: c20H32O3
Molecular weight: 320.24
The characteristics are as follows: colorless crystals
Optically active
IR(KBr)vmax:3421,2929,1629,1459,1381,1040,635cm-1。
UV/Vis(MeOH)λmax(logε):205(3.83)nm。
EIMS[M+Na]+m/z:343。
HRESIMS[M+Na]+m/z:343.2243(calcd for 343.2249)。
The molecular formula is as follows: c20H32O3
Molecular weight: 320.24
The characteristics are as follows: colorless crystals
Optically active
IR(KBr)vmax:3423,2925,1630,1464,1382,1050cm-1。
UV/Vis(MeOH)λmax(logε):203(3.57)nm。
EIMS[M+Na]+m/z:343。
HRESIMS[M+Na]+m/z:343.2246(calcd for 343.2249)。
The molecular formula is as follows: c20H34O3
Molecular weight: 322.25
The characteristics are as follows: colorless crystals
Optically active
IR(KBr)vmax:3374,2929,1631,1384,1069,878,699cm-1。
UV/Vis(MeOH)λmax(logε):206(3.92)nm。
EIMS[M-H]-m/z:321。
HRESIMS[M+Na]+m/z:345.2402(calcd for 345.2406)。
Example 2:
isopimarane diterpene derivatives (1-7) were prepared according to the method of example 1, and were dissolved in a small amount of DMSO, and then injection water was added thereto, followed by fine filtration, potting and sterilization, to prepare an injection.
Example 3:
the isopimarane diterpene derivatives (1-7) were prepared according to the method of example 1, and after dissolving in a small amount of DMSO, dissolved in sterile water for injection, stirred to dissolve, filtered with a sterile suction filter funnel, then sterile fine filtered, subpackaged in ampoules, freeze-dried at low temperature and then sterile melt-sealed to obtain powder for injection.
Example 4:
adding excipient into the separated isohimarane diterpene derivatives (1-7) according to the weight ratio of the isohimarane diterpene derivatives to the excipient of 9:1, and making into powder.
Example 5:
the isopimarane diterpene derivatives (1-7) were prepared according to the method of example 1, and the excipients were added in a weight ratio of 5:1 to the excipients, respectively, and granulated and tabletted.
Example 6:
isopimarane diterpene derivatives (1-7) were prepared according to the method of example 1, and oral liquids were prepared according to the conventional oral liquid preparation methods, respectively.
Example 7:
the isopimarane diterpene derivatives (1-7) were prepared according to the method of example 1, and the excipients were added in a weight ratio of 5:1 to the excipients, respectively, to prepare capsules.
Example 8:
the isopimarane diterpene derivatives (1-7) were prepared according to the method of example 1, and the excipients were added in a weight ratio of 3:1 to the excipients, respectively, to prepare capsules.
Claims (8)
1. Isopimarane diterpene derivatives 2-3 shown in structural formula (I),
2. a pharmaceutical composition comprising a therapeutically effective amount of any one of the isopimarane-type diterpene derivatives 2-3 of the formula (I) described in claim 1 and a pharmaceutically acceptable carrier.
3. Use of the isopimarane-type diterpene derivatives 2-3 of claim 1 in the preparation of estrogen biosynthesis regulators.
4. Use of the isopimarane-type diterpene derivatives 2-3 of claim 1 in the preparation of medicaments for treating various diseases mediated by estrogen.
5. Use of the pharmaceutical composition of claim 2 for the preparation of an estrogen biosynthesis modulator.
6. Use of the pharmaceutical composition of claim 2 for the manufacture of a medicament for the treatment of a variety of estrogen-mediated conditions.
7. The process for producing the isopimarane-type diterpene derivatives 2 to 3 of the formula (I) according to claim 1, characterized by taking the leaf and branch parts of premna fulva, drying, pulverizing, extracting with 80% ethanol at room temperature for 3 times, combining the ethanol extracts, filtering, concentrating under reduced pressure; suspending the extractive solution in water solution, extracting with ethyl acetate, concentrating the ethyl acetate part under reduced pressure, dissolving with chloroform, adsorbing on silica gel, standing at room temperature for volatilizing solvent, performing silica gel column chromatography, and gradient eluting with chloroform/acetone of 1:0 → 0:1 to obtain five parts A-E; wherein the C part is decolorized with MCI, eluted with 95% methanol/water, concentrated, and then separated again with RP-18 reverse phase silica gel, and eluted with methanol/water gradient to obtain C1-C8 fraction; c4 is further separated by Sephadex LH-20 gel, and is eluted by methanol to obtain C4.1-C4.5 fraction; separating the C4.3 part with normal phase silica gel, eluting with petroleum ether/acetone at 8:1-1:1 gradient, separating into C4.3.1-C4.3.8 fractions, recrystallizing derivative 2 from C4.3.5 fraction, and further purifying fraction C4.3.4 with HPLC, acetonitrile/water at 48:52, 3mL/min to obtain derivative 3.
8. The process for preparing the pharmaceutical composition according to claim 2, characterized in that the leaf and branch parts of the tofu padding are taken, dried, pulverized, extracted with 80% ethanol at room temperature for 3 times, the ethanol extracts are combined, filtered and concentrated under reduced pressure; suspending the extractive solution in water solution, extracting with ethyl acetate, concentrating the ethyl acetate part under reduced pressure, dissolving with chloroform, adsorbing on silica gel, standing at room temperature for volatilizing solvent, performing silica gel column chromatography, and gradient eluting with chloroform/acetone of 1:0 → 0:1 to obtain five parts A-E; wherein the C part is decolorized with MCI, eluted with 95% methanol/water, concentrated, and then separated again with RP-18 reverse phase silica gel, and eluted with methanol/water gradient to obtain C1-C8 fraction; c4 is further separated by Sephadex LH-20 gel, and is eluted by methanol to obtain C4.1-C4.5 fraction; separating the C4.3 part by normal phase silica gel, eluting with petroleum ether/acetone at a gradient of 8:1-1:1, separating into C4.3.1-C4.3.8 fractions, recrystallizing the derivative 2 from C4.3.5 fraction, and further purifying the fraction C4.3.4 by HPLC, acetonitrile/water at 48:52, 3mL/min to obtain derivative 3; adding the isopimarane diterpene derivatives 2-3 obtained in the steps into a medicinal carrier respectively to obtain the medicinal composition.
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| CN105198951A (en) * | 2015-10-22 | 2015-12-30 | 云南民族大学 | Tetracyclic diterpenoid compound and preparation method as well as application thereof |
| CN108129295A (en) * | 2018-01-12 | 2018-06-08 | 云南大学 | A kind of Diterpene derivative and its pharmaceutical composition and purposes |
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| CN108129295A (en) * | 2018-01-12 | 2018-06-08 | 云南大学 | A kind of Diterpene derivative and its pharmaceutical composition and purposes |
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| Cytotoxic Diterpenoids from the Roots of Aralia melanocarpa;Zhi-Yong Jiang等;《Planta Medica》;20160227;第82卷(第8期);742-746 * |
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